Short-hairpin RNAs delivered by lentiviral vector transduction trigger

Short-hairpin RNAs delivered by lentiviral vectortransduction trigger RIG-I-mediated IFN activationRachael Kenworthy1, Diana Lambert1, Feng Yang1, Nan Wang2, Zihong Chen3,

Haizhen Zhu4, Fanxiu Zhu1, Chen Liu4, Kui Li2 and Hengli Tang1,*

1Department of Biological Science, Florida State University, Tallahassee, FL 32306-4295, 2Department ofMolecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, 3Department ofMicrobiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555 and 4Departmentof Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL 32611, USA

Received May 15, 2009; Revised August 13, 2009; Accepted August 14, 2009

ABSTRACT

Activation of the type I interferon (IFN) pathway bysmall interfering RNA (siRNA) is a major contributorto the off-target effects of RNA interference inmammalian cells. While IFN induction complicatesgene function studies, immunostimulation bysiRNAs may be beneficial in certain therapeuticsettings. Various forms of siRNA, meeting differentcompositional and structural requirements, havebeen reported to trigger IFN activation. Theconsensus is that intracellularly expressed short-hairpin RNAs (shRNAs) are less prone to IFNactivation because they are not detected bythe cell-surface receptors. In particular, lentiviralvector-mediated transduction of shRNAs has beenreported to avoid IFN response. Here we identifya shRNA that potently activates the IFN pathway inhuman cells in a sequence- and 50-triphosphate-dependent manner. In addition to suppressing itsintended mRNA target, expression of the shRNAresults in dimerization of interferon regulatoryfactor-3, activation of IFN promoters and secretionof biologically active IFNs into the extracellularmedium. Delivery by lentiviral vector transductiondid not avoid IFN activation by this and another,unrelated shRNA. We also demonstrated thatretinoic-acid-inducible gene I, and not melanomadifferentiation associated gene 5 or toll-like recep-tor 3, is the cytoplasmic sensor for intracellularlyexpressed shRNAs that trigger IFN activation.

INTRODUCTION

A specific double-stranded RNA (dsRNA) structure,�21–22 bp dsRNA with 30 overhangs, plays a critical

role in initiating both microRNA (miRNA)- and smallinterfering RNA (siRNA)-mediated gene silencing, as itis the structure recognized by the RNA interference(RNAi) machinery, the RNA-induced silencing complex(RISC) (1–3). Except for preformed siRNA duplexes of�21 bp, the RISC-loaded small RNAs are generated by aribonuclease (RNase) III-like enzyme that is found invirtually all eukaryotic organisms. This enzyme, aptlynamed Dicer for its ability to cleave a variety of larger(>30 bp) dsRNA molecules into the �21 bp dsRNA witha characteristic 30 overhang of 2 nt, is a multidomainRNA-binding protein and itself a component of RISC.The primary sequence of the RNAs is not important inRISC formation, and RNAi can suppress virtually anytarget as long as rules of sequence complementaritiesbetween the small RNA and the target RNA are satisfied.dsRNAs are also a type of pathogen-associated

molecular pattern (PAMP) that are detected by cellularinnate immunity sensors named Pattern RecognitionReceptors (PRRs) (4). The interaction between a PAMPand a PRR triggers activation of the interferon (IFN)pathway in mammalian cells, which significantly changesthe gene-expression profile in the cells and contributes tothe well-documented off-target effect of RNAi. IFNinduction is especially problematic in antiviral studiesemploying RNAi, where the antiviral effect of IFN mustbe distinguished from that of RNAi.Typical IFN-inducing structure patterns include

dsRNA of certain length, single-stranded RNA (ssRNA)containing 50-triphosphates (50-ppp), the dsRNA analoguepolyinosinic-polycytidylic acid (poly I:C), and certaindsDNA molecules. These RNA patterns are generallybelieved to possess ‘non-self’ properties to allow the cellto recognize foreign (often viral) RNAs specifically.Various forms of siRNA duplexes have been reported totrigger IFN induction both in vitro and in vivo (5–9),probably through the cell surface- and/or endosome-expressed Toll-like receptors (TLRs), including TLR3and TLR7 (6,8,9).

*To whom correspondence should be addressed. Tel: +1 850 645 2402; Fax: +1 850 645 8447; Email: [email protected]

Published online 3 September 2009 Nucleic Acids Research, 2009, Vol. 37, No. 19 6587–6599doi:10.1093/nar/gkp714

� The Author(s) 2009. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Short-hairpin RNAs (shRNAs) expressed from a DNAplasmid have also been shown to activate IFN (10). Thedouble-stranded form of these RNAs is below the sizelimit of the stem-loop RNAs that can be detected bythe RNA-activated protein kinase (PKR) (11) and isprobably detected by other cytoplasmic PRRs. Twocytoplasmic RNA helicases, retinoic-acid-inducible geneI (RIG-I) and melanoma differentiation associatedgene 5 (MDA5), signal to the IFN-b promoter whenactivated by specific RNA structures (12–14). Althoughboth PRRs signal through the mitochondrial antiviralsignaling protein MAVS/Cardif/VISA/IPS-1 (15–18),studies of ligand specificity suggest that RIG-I andMDA5 are parallel sensors with overlapping substrates.For example, although both PRRs are activated by polyI:C in cell culture systems (12,19–23), MDA5 appears tobe more important in mediating the poly I:C response invivo (13,14). In addition, RIG-I can bind and respond tossRNAs bearing 50-ppp, whereas MDA5 is not activatedby 50-ppp–containing RNA (24,25). Finally, severalcytosolic sensors for dsDNA has been recently reported(26–31). Nevertheless, current data on what constituteseffective substrates for either PRR are incomplete andsometimes controversial. Here we report for the firsttime that shRNAs delivered by lentiviral transductiontriggered IFN activation and that RIG-I and MAVS,but not MDA5 or TLR3, mediated the IFN activationtriggered by intracellularly expressed shRNA, whichcould activate both IFN-a and IFN-b promoters. IFNactivation depended on sequence, a 50-ppp and correctprocessing of the RNA hairpin by Dicer; it wasindependent of promoter choice, presence of blunt ends,route of delivery and RNAi potency.

MATERIALS AND METHODS

Cells, antibodies and RNAs

GS5 and LH86 cells have been described earlier (32,33).Huh-7 and 293FT cells were maintained in DMEM supple-mented with 10% FBS. We used the following antibodies:anti-CyPA (Biomol, Plymouth Meeting, PA, USA); anti-CyPB (AfEnity BioReagents, Rockford, IL, USA); anti-Ku80, anti-Flag and anti-actin (Sigma-Aldrich, St Louis,MO, USA); anti-IFN stimulate gene (ISG)15 (RocklandImmunochemicals, Gilbertsville, PA, USA); anti-NS5A(Virogen, Watertown, MA, USA) and anti-NS3(in-house). GSB1 and H801 cells have been describedearlier (34). Poly I : C was purchased from Sigma-Aldrich,and synthetic hairpin RNAwas purchased from IntegratedDNA Technologies (Coralville, IA, USA). SyntheticsiRNA was purchased from Ambion (Austin, TX, USA).

Gel electrophoresis and western blotting

Protein contents of cell lysate were quantified with theBio-Rad DC protein assay (Bio-Rad, Hercules, CA,USA), and an equal amount of total protein was loadedin each lane. Samples for IRF-3 dimerization assay wererun on a polyacrylamide gel under non-denaturingconditions (35). Other samples were denatured andseparated by sodium dodecyl sulfate polyacrylamide

gelelectrophoresis (SDS–PAGE). Proteins were thentransferred onto a nitrocellulose membrane and stainedwith the appropriate antibodies with the SNAP i.d.TM

system (Millipore, Worcester, MA, USA) according tothe manufacturer’s instructions.

Transfections

For luciferase assays, cells were seeded to a confluencyof 50%, and for all other assays, cells were seeded toa confluency of 30%. The next day, transfections ofDNA plasmids and synthetic RNAs were performedwith LipofectamineTM 2000 (Invitrogen, Carlsbad, CA,USA) according to the manufacturer’s instructions.

Plasmids

Plasmids pGL3-IFNA1, pGL3-IFNB, pRL-TK, pCMV-Flag-IRF-3 and pCR3.1-IRF-7A have been describedearlier (36). shRNAs were expressed from a humanimmunodeEciency virus (HIV)-based lentiviral vector(32,37), and sh-PCAF was constructed on the basis of apreviously reported sequence (38). Plasmid sh-B971/H1was constructed by cloning of the DNA fragmentencoding the sh-B971 RNA into pSilencer 3.0-H1(Ambion, Austin, TX, USA) according to the manu-facturer’s instructions. The RIG-I and TLR3 constructshave been described (39,40). The RIG-I C constructencodes Flag-tagged, C-terminal 707 aa of human RIG-Icloned into a bicistronic expression vector modified frompBICEP-CMV-1 (Sigma-Aldrich, St Louis, MO, USA), inwhich the CMV promoter was replaced with theelongation-factor-1 promoter. The MDA5, MDA5-Cconstructs were kindly provided by Fujita (12). HCVgenotype 2a NS3-4A protease was expressed from thepCMV-3Tag-1a plasmid (Stratagene, La Jolla, CA, USA).

Luciferase assay for Interferon promoter activity

293FT cells were seeded in 24-well plates and weretransfected 16 h later with 400 ng of a shRNA expressionvector, 40 ng of pGL3-IFNA1 or pGL3-IFNB, 20 ng ofpRL-TK and 50 ng of pCR3.1-IRF-7A. Cells werecollected 48 h after transfection. Luciferase assays wereperformed with the Dual-Glo� Luciferase Assay systemreagents (Promega, Madison, WI) and luminescencequantified with a Modulus Microplate reader (TurnerBioSystems, Sunnyvale, CA, USA). Ratios of fireflyluciferase (from the pGL3 vectors) to Renilla luciferase(from the pRL-TK vector) were calculated, and that ofthe sh-B971 sample was normalized to 100%.

Lentiviral vectors

Sequences of shRNA are shown in Table 1. Lentiviralvector production and transduction were performed asdescribed earlier (37). Viral vectors were pelleted byultracentrifugation at 50 000g at 4�C for 3 h andresuspended in a volume of PBS that was 1% of theoriginal medium volume. The titers of the concentratedvectors were then measured with a p24 ELISA kit(ZeptoMetrix, Buffalo, NY, USA).

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Real-time reverse transcription PCR

Real-time reverse transcription PCR (RT–PCR) wasperformed as described earlier (32). The primers usedwere OAS1 forward, 50-AGG TGG TAA AGG GTGGCT CC-30 and OAS1 reverse 50-ACA ACC AGG TCAGCG TCA GAT-30; RIG-I forward 50-GAG GCA GAGGAA GAG CAA GAG G-30 and RIG-I reverse 50-CGCCTT CAG ACA TGG GAC GAA G-30; GAPDHforward 50-TCA CTG CCA CCC AGA AGA CTG-30

and GAPDH reverse 50-GGA TGA CCT TGC CCACAG C-30. The primers for HCV detection were 50-CGCTCA ATG CCT GGA GAT TTG-30 and 50-GCA CTCGCA AGC ACC CTA TC-30.

Flow cytometry

For flow cytometry, GS5 cells were fixed 48 h aftertreatment in a solution of 2% paraformaldehyde andanalyzed with a FACSCanto flow cytometer (BDBiosciences, San Jose, CA, USA). Mean GFP intensitywas plotted, and that of the sh-NTC sample wasnormalized to 100%.

RNA extraction and northern blots

Total RNA from transiently transfected 293FT cells wasextracted with RNA STAT-60 (Tel-Test, Friendswood,TX, USA) and separated on a 7.5% urea polyacrylamidegel. The transfer of RNA onto nitrocellulose membraneand hybridization were performed according to standardmolecular biology protocols. The probe for detecting theexpression of sh-B971 and its variants was a synthetic DNAoligomer corresponding to the bottom strand of sh-B971.Radioactive labeling of the probe was performed withan end-labeling protocol with T7 polynucleotide kinase(Ambion, Austin, TX, USA). The exposure and detectionof the radioactive signal was performed with a TyphoonImager (GE Healthcare, Piscataway, NJ, USA) withQuantity One software (Bio-Rad, Hercules, CA, USA).

RESULTS

A short-hairpin RNA directed at CyPB induces IFNproduction in human embryonic kidney cells

To investigate the potential role of the cyclophilins (CyPs)in HCV replication (41), we delivered several shRNAsdirected at mRNAs of three CyPs into HCV repliconcells by means of a lentiviral vector, using a murineU6 promoter to drive the expression of the shRNA(Figure 1A) (37). We observed a discrepancy betweentwo anti-CyPB shRNAs (B971 and B710) in theirrelative efficiency in knocking down CyPB expressionand in suppressing HCV. Lentiviral vector sh-B971 wasless efficient in knocking down CyPB expression butpotently inhibited HCV NS5A expression in a humanhepatoma cell line containing replicating HCV RNA(Figure 1B, left). Viral inhibition was independent ofCyPB knockdown, as control medium from transfected293FT cells that did not contain any lentiviral vectorparticles, generated by omission of the packagingplasmids during transfection, also inhibited HCV

replication (Figure 1B, right) without affecting CyPBexpression. The fast kinetics of viral inhibition (completeinhibition with 48 h, data not shown) was also more con-sistent with IFN than with RNAi-based inhibition. Thepresence of IFN in the lentiviral vector preparation ofsh-B971 was confirmed by strong induction of 20-50-oligo-adenylate synthetase 1 (OAS1), a classic IFN-inducedgene, in both naı̈ve Huh-7 and the HCV replicon cellline (GS5) treated with the medium (Figure 1C). Inaddition, HCV replication in an IFN-resistant HCVreplicon cell line (H801), in contrast to that in a wild-type replicon cell line (GSB1) (34), was not inhibited bythe sh-B971 medium (Figure 1D), suggesting the lack ofadditional viral inhibiting agents in the sh-B971 medium.Expression of sh-B971 in 293FT cells also induceddimerization of IRF-3, confirming the activation of theIFN production pathway in these transfected cells(Figure 1E). Finally, sh-B971 was able to activate bothIFN-a and IFN-b promoters, although the activationof the IFN-a promoter required coexpression of IRF-7,which is normally expressed at very low levels in 293-basedcells (Figure 1F). These results demonstrate that sh-B971is a potent activator of IRF-3 and IRF-7, masterregulators of IFN expression in human cells.

RIG-I mediates the IFN induction by sh-B971

We next investigated the role of the different viral/exogenous RNA sensors, RIG-I, MDA5 and TLR3,in sh-B971–triggered IFN production. Mammalianexpression plasmids encoding each of these proteins, aswell as the dominant negative (DN) mutants of RIG-Iand MDA5, were transfected into 293FT cells withshRNAs and an IFN-b promoter reporter construct.The signaling to IFN-b promoter and the expression ofthe PRR proteins were then examined 48 h aftertransfection. In the absence of sensor proteins, the sh-B971 increased activation of the IFN-b promoter by2.6-fold (Figure 2A). Coexpression of MDA5 or TLR3did not increase or decrease sh-B971’s ability to activateIFN-b promoter relatively to the negative control shRNA(sh-NTC), but in the presence of RIG-I coexpression, theinduction of IFN-b promoter by sh-B971 was increased to�30-fold. Moreover, ectopic expression of a DN mutantof RIG-I (RIG-I C), but not that of MDA5 (MDA5-C),completely abrogated IFN promoter activation bysh-B971. With the exception of TLR3, which requiredprolonged exposure of the western blot to be detected,the cytoplasmic sensors and their mutants were expressedat comparable levels (Figure 2B). Moreover, activation ofIRF-3 (Figure 1E) and IFN promoters (Figure 1F) in293FT cells, which do not contain a functional TLR3signaling pathway (42), indicates that TLR3 plays anegligible role, if any, in IFN induction by sh-B971.The combination of sh-B971 and RIG-I produced thehighest level of IFN-b promoter activity, which wereconfirmed by western blotting showing that endogenousISG15 induction was only detectable in cells cotransfectedwith sh-B971 and wild-type RIG-I (Figure 2B). Toconfirm further that biologically active IFN was releasedfrom these cells, we applied the culture medium of the

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transfected 293FT cells to an HCV replicon cell line (GS5)in which NS5A-GFP expression is used for monitoringviral RNA replication (43). HCV replication in this cellline is extremely sensitive to IFN, and the effect of the

cytokine can be readily measured as the change in themean GFP intensity of the treated cells. As shown inFigure 2C, culture medium from sh-B971 efficientlysuppressed HCV replication, resulting in a decrease in

Figure 1. A small-hairpin RNA directed at CyPB induces IFN production in human embryonic kidney cells. (A) Sequence of sh-B971, which wasexpressed from a self-inactivating human immunodeficiency virus (HIV) vector with a murine U6 promoter (59). (B) Inhibition of HCV expression byculture media of sh-B971-transfected 293FT cells. GS5 cells were treated with culture supernatant taken from 293FT cells transfected with variousshRNA plasmids with (left) or without (right) the packaging plasmids overnight. Cells were then cultured in fresh media for an additional 6 daysbefore being lysed for western blotting. (C) OAS1 induction by culture supernatant from 293FT cells transfected with sh-B971. Huh 7 and GS5 cellswere treated with culture supernatant from 293FT cells transfected with either sh-Luc or sh-B971 for 24 h before RNA extraction and real-time RT–PCR analysis. OAS1 RNA level was normalized to that of GAPDH RNA. (D) Transfected culture media failed to suppress HCV replication in anIFN-resistant cell line. HCV replicon cells were cultured as described earlier (34) and then treated with the indicated culture medium from transfected293FT cells. HCV RNA was analyzed with real-time RT–PCR. (E) IRF-3 dimerization in response to sh-B971 expression. Flag-IRF-3 wascotransfected with a shRNA into 293FT cells. Cells were lysed 24 h after transfection, and total cell lysate was separated on a polyacrylamidegel under non-denaturing conditions, transferred and stained with an anti-flag antibody. (F) IFN-a and IFN-b promoter activation by sh-B971expression. Sh-NTC, sh-C454 (an shRNA directed at CyPC), or sh-B971 was cotransfected along with luciferase reporter plasmids with or withoutIRF-7. The ratios of firefly luciferase readings to Renilla luciferase readings were plotted.



the NS5A-GFP intensity within 48 h of treatment.Cotransfecting wild-type RIG-I produced a mediumwith stronger inhibition, whereas the RIG-C drasticallysuppressed the antiviral effect of the medium. Finally,real-time RT–PCR analysis revealed that sh-B971, butnot the negative control shRNA, strongly activatedexpression of endogenous RIG-I, a well-characterizedISG whose induction requires paracrine/autocrine actionof IFN (44,45). As expected, poly I : C activated RIG-Iexpression in the same assay (Figure 2D). These results,taken together, show that RIG-I is the cellular sensor thatmediates the IFN induction by sh-B971.

Structure and sequence determinants of shRNA-mediatedIFN activation

The majority of the shRNAs that we use in the lab do notactivate RIG-I expression and IFN signaling despitehaving essentially the same structure as sh-B971, so wewanted to determine whether the sequence of sh-B971is distinctive enough to trigger the production of IFN.We first tested a synthetic siRNA duplex with the sametarget sequence as sh-B971. This siRNA (si-B971-syn)should resemble the final Dicer product of sh-B971except for the 50-ends. The synthetic siRNA contains50-OH groups, whereas the Dicer products probably

Figure 2. Sh-B971 acts through the RIG-I pathway to trigger IFN activation. (A) RIG-I, and not melanoma differentiation associated gene 5(MDA5) or toll-like receptor 3 (TLR3), mediated IFN induction by sh-B971. Various PRRs and their mutant proteins were coexpressed with eithersh-NTC or sh-B971 along with the luciferase reporters. The firefly luciferase readings were normalized to Renilla luciferase readings, and the value ofsh-B971 was set to 100. (B) Proper expression of the transfected PRR proteins and induction of ISG 15 expression when sh-B971 was coexpressedwith RIG-I. RIG-I, RIG-I C, MDA5 and MDA5 C are all tagged with two tandem copies of Flag epitope. TLR3 is tagged with single Flag epitopeand required prolonged exposure for detection. (C) A dominant negative mutant of RIG-I blocked sh-B971-triggered IFN production. ShRNAs werecotransfected with either RIG-I or a dominant negative form of RIG-I (RIG-I C) into 293FT cells. The culture supernatant was then tested for itsability to suppress NS5A-GFP expression in GS5 cells with an overnight treatment. (D) Upregulation of RIG-I RNA level by sh-B971 expression.293FT cells were transfected with sh-NTC, sh-B971 or polyinosinic-polycytidylic acid. RNA was extracted 24 h after transfection and analyzed byreal-time RT PCR. The RIG-I RNA levels were normalized to those of GAPDH RNA.



contain a 50-monophosphate on one strand and a50-triphosphate on the other. Si-B971-syn knocked downCyPB expression as efficiently as sh-B971 (Figure 3A)while failing to activate IFN production, as measured bythe GFP-HCV assay (Figure 3B). To determine whetherthe sequence of the intact hairpin RNA before Dicercleavage is sufficient to trigger IFN, we tested a syntheticshRNA (sh-B971-syn) that had exactly the same sequenceas the predicted intracellular sh-B971 transcript generatedby the U6 promoter. Again, the 50-end of the syntheticsh-B971 had a 50-OH group instead of any phosphate.Sh-B971-syn behaved similarly to si-B971-syn in that itknocked down CyPB expression without activatingIFN response (Figure 3). These results suggest that the50-end status of sh-B971 is important for IFN activation,consistent with the previously finding that a50-triphosphate is required for RIG-I activation (24,25).To determine the contribution of the individual residues

of the sh-B971 sequence, we introduced a series ofpoint mutations into the shRNA and tested them forIFN induction. We changed the first nucleotide fromA to G, C, or T while maintaining base-pairing betweennucleotides +1 and +47. These mutant shRNAs lackedthe ability to activate IFN production (Table 1). Changingthe +1 nucleotide to G while leaving the +47 nucleotideintact also abolished IFN activation by the shRNA(A1/G), as did the reciprocal mutation U47/C. Theimportance of the first nucleotide was further confirmedby the inability of sh-B971+1 to activate IFN. The targetof sh-B971+1 was shifted 1 nt downstream on the CyPBmRNA, producing an shRNA starting with a G at the +1position. The presence of an A at the +1 position was not,however, sufficient to render a shRNA competent for IFNactivation, as replacing the first nucleotide of the sh-NTCwith an A did not generate an IFN-inducing shRNA(NTC-A and NTC+1). These results indicate that aprotruding/unpaired A at the end of the hairpin or theRNA duplex, a potential result of ‘breathing’ at the endof the dsRNA, is not sufficient to trigger IFN induction aspreviously suggested (38).Two point mutations located farther into the stem

structure of the shRNA (9G9 and B18A1) also reduced

its ability to induce IFN even though the base-pairing wasperfectly maintained in these mutants. Finally, replacingthe 9-nt hairpin loop with a 7-nt loop that had beenpreviously shown to abolish shRNA-mediated RNAi(loop A mutant) (46) eliminated sh-B971’s ability to

Figure 3. Structural determinants of IFN activation by sh-B971. 293FT cells were transfected with shRNA-expressing lentiviral plasmids or syntheticRNAs. (A) Knockdown of CyPB expression by various forms of B971 siRNA. Cells were collected 5 days after transfection and analyzed by westernblot for detection of CyPB. (B) Lack of HCV inhibition by synthetic forms of sh-B971. Culture supernatant from transfected 293FT cells wascollected 48 h after transfection and used to treat GS5 cells overnight; the GS5 cells were then analyzed by flow cytometry.

Table 1. Sequence of the siRNA duplexes



induce IFN, suggesting the importance of RNAprocessing in the induction. To determine whether theinability of the mutant shRNAs to induce IFN was dueto lower expression levels, we performed northern blottinganalysis of the shRNA expression on the wild-type andtwo mutants. The mutants A1/G and Loop A werechosen because their final siRNA products have exactlythe same sequence as that of the wild-type sh-B971 andcan thus be detected with the same efficiency by the sameprobe. Although sh-A/G and sh-Loop A were clearlyunable to activate IFN-b promoter (Figure 4A), theywere both expressed at levels comparable to those of thewild-type sh-B971 product (Figure 4B). Interestingly, thefinal siRNA product of sh-Loop A was slightly smallerthan those of sh-B971 and sh-A1/G, suggesting thatcleavage did occur and perhaps occurred one or 2 nt intothe stem to compensate for the shorter loop.

RIG-I-mediated IFN induction by sh-B971 is independentof a blunt end of the dsRNA

Blunt-ended siRNA has been previously reported to bestronger inducers of IFN than the siRNAs with overhangs(47). Indeed, a previously reported IFN-inducing shRNA,sh-PCAF (p300/CREB-binding protein-associated factor),contains a blunt end (38) and was more potent inactivating IFN than sh-B971 (Figure 5A), which ispredicted to form an overhang of 2–3Ts at each end of

the final siRNA. We therefore constructed a version of thesh-B971 that would be blunt at the end that is notprocessed by Dicer by adding two extra As to the 50-endof the shRNA. This modification (Blunt sh-B971) didnot increase the ability of sh-B971 to activate IFN-bpromoter (Figure 5A). We confirmed, in two independentexperiments, that IFN induction by sh-PCAF was alsomediated by RIG-I. First, cotransfection of DN RIG-Iresulted a 50- to 100-fold inhibition of IFN induction bysh-PCAF (Figure 5B), whereas wild-type RIG-I increasedIFN induction by several fold in the same assay. Second,when HCV NS3-4A protease, which cleaves MAVS,thereby blocking the RIG-I pathway, was coexpressedwith either sh-B971 or sh-PCAF, IFN induction by theseshRNAs were severely compromised (Figure 5C), furthersubstantiating a role of the RIG-I and MAVS pathwayin mediating IFN induction by both the blunt-endedsh-PCAF and the sh-B971 with overhang. The properexpression of NS3-4A protease was confirmed bywestern blotting (Figure 5D).

Sh-B971 expressed from an H1 promoter triggers IFNinduction

To assess the contribution of the promoter choice in IFNactivation by intracellular expressed shRNA, we expressedsh-B971 from another commonly used pol III promoter,the human H1 promoter. Both the original, mU6-drivensh-B971 and the H1-driven sh-B971 activated IFN-bpromoter (Figure 6A) and resulted in secretion of IFNinto the transfected cell-culture media, which in turnsuppressed HCV replication (Figure 6B). Properexpression of the siRNA (Figure 6C) and the subsequentknockdown of CyPB expression (Figure 6D) all appearednormal for sh-B971 expressed from the H1 promoterplasmid, which has a backbone different from that ofour lentiviral vector carrying the mU6 promoter. Thesedata suggest that IFN induction by sh-B971 is notrestricted to a particular promoter or expressionconstruct. Further supporting this conclusion was theobservation that the expression cassette by itself,removed and isolated from the lentiviral plasmid byrestriction digestion, could also activate IFN productionin transfected 293FT cells (data not shown).

ShRNAs delivered via lentiviral transduction trigger IFNactivation in vitro

To this point, all the IFN induction experiments weredone with transient transfection of DNA vectors and itwas possible that certain features of the double-strandedplasmid DNA are responsible for IFN induction. We firsttried to address this point by transfecting just the shRNA-expressing cassette, generated either by PCR or restrictionenzyme digestion, into 293FT cells and confirming thatthese fragments of �200 bp were sufficient to triggerIFN induction (Supplementary Figure S1). To definitivelyrule out any contribution by dsDNA, we used a lentiviraltransduction system which has been suggested to expressshRNAs that can escape detection by PRRs and IFNactivation (48). We produced lentiviral particlescontaining shRNAs from 293FT cells using standard

Figure 4. Comparably expressed mutant forms of sh-B971 do notinduce IFN. (A) IFN activation by select sh-B971 mutants. TheshRNA expression plasmids were transfected into 293FT cells withthe luciferase reporters to measure IFN-b promoter activation. Thefirefly luciferase readings were normalized to Renilla luciferasereadings, and the value of sh-B971 was set to 100. (B) Intracellularlevels of siRNA products of sh-B971 and mutants. RNA was extractedfrom transfected 293FT cells and analyzed by northern blotting with aDNA oligonucleotide probe that is complementary to all three forms ofsh-B971 (wt, sh-A1/G and Loop A).



Figure 5. RIG-I mediated IFN induction by shRNA is independent of a blunt end of the dsRNA. (A) IFN-b activation in response to blunt-endedshRNAs. Both sh-PCAF and Blunt sh-B971 contained two extra As at the 50-end of the shRNA, making the non-hairpin end of the shRNA bluntrather than having an overhang of two TTs. (B) RIG-I dependency of IFN-b activation by sh-PCAF. (C) Blockade of shRNAs-triggered IFNactivation by HCV NS3-4A. A mammalian expression plasmid encoding the NS3-4A protease from HCV isolate JFH-1 was cotransfected with theshRNA and luciferase reporter plasmids. (D) Western blot showing expression of HCV NS3-4A in transfected 293FT cells. Cell lysate from (C) wasseparated by SDS–PAGE and probed with an anti-NS3 antibody.

Figure 6. Sh-B971 expressed from an H1 promoter triggers IFN activation. Sh-B971 expressed from an H1 promoter was capable of (A) activatingIFN-b promoter and (B) triggering IFN production to inhibit HCV replication in GS5 cells. (C) Intracellular levels of U6- and H1-driven sh-B971products. RNA extraction and northern blotting were performed as described in Figure 4B. (D) Knockdown of CyPB expression by sh-B971expressed from an H1 promoter.



methods, centrifuged them to separate the vectors fromthe IFN-containing media, and then used them to infectnaı̈ve 293FT cells (Figure 7A). Both sh-B971 andsh-PCAF vectors induced IFN production when deliveredas concentrated lentiviral particles, measured both by

HCV suppression (Figure 7B) and by OAS induction(Figure 7C) in Huh-7 cells. To rule out the possibilitythat residual IFN in the concentrated viral particles wasresponsible for these results, we added 100U/mlIFN to the negative control vector sample before the

Figure 7. ShRNAs delivered by lentiviral transduction trigger IFN activation in vitro. (A) Diagram of the experimental setup. Lentiviral vectors wereconcentrated by ultracentrifugation for removal of soluble IFN proteins in the vectors prepared from transfected 293FT cells. (B) Transduction of293FT cells with the concentrated shRNA vectors triggered IFN production. Culture supernatant was collected 48 h after transduction and was usedto treat GS5 cells overnight; the GS5 cells were then analyzed 48 h after treatment by flow cytometry. (C) OAS induction in Huh 7 cells treated withculture supernatant from lentivirally transduced cells. Huh 7 cells were treated with culture supernatant for 24 h before RNA extraction and RT-PCRanalysis. OAS1 expression level was normalized to that of GAPDH RNA. (D) Knockdown of CyPB expression in transduced cells, which werecollected 5 days after transduction for western blotting. (E) Blockade of lentivirus-triggered IFN activation by a HIV reverse transcriptase inhibitor.293FT cells were transduced in the presence of 80 nM of Nevirapine; culture supernatant was collected 48 h after transduction and used to treat GS5cells. (F) Culture medium from LH86 cells transduced with sh-PCAF suppressed HCV replication. This experiment was done as depicted in (A) withthe LH86 cells in the place of 293FT cells in the transduction step.



concentration step. This preparation, designatedsh-NTC*, was not able to trigger IFN production innaı̈ve 293FT cells, suggesting that the concentration stepeffectively removed the soluble IFN from the viral particlepellet. Proper knockdown of the siRNA target of sh-B971was confirmed by this route of shRNA delivery (Figure7D). To prove definitively that IFN induction by theshRNAs was mediated by the lentiviral infection route,we tested the effect of an inhibitor of HIV reversetranscriptase, Nevirapine, on IFN induction by sh-B971and sh-PCAF. As shown in Figure 7E, inclusion ofNevirapine at the time of transduction effectivelyblocked the ability of both shRNAs to induce IFN inthe transduced cells, suggesting the importance of thereverse transcription step in the expression of theshRNAs delivered by the lentiviruses. To determinewhether lentiviral vector-delivered shRNA can triggerIFN induction in cells other than 293FT cells, wetransduced a human hepatoma cell line, LH86, whichhas been reported to produce IFN upon viral infection(33), and examined IFN induction in these cells. Culturemedium from LH86 cells transduced with sh-PCAFcontained biologically active IFN, which suppressedHCV replication in GS5 cells (Figure 7F), indicatingthat the ability of shRNAs delivered by lentivirus toinduce IFN response was not limited to 293FT cells.

DISCUSSION

It has been reported that certain chemically synthesizedand phage polymerase in vitro transcribed siRNAs cannon-specifically induce IFN responses and produce off-target effect via various PRRs, including TLRs.However, the induction of IFN response by shRNAsand its underlying mechanisms have not been as wellstudied. The actual number of shRNAs that are capableof triggering IFN response will certainly be larger than thefew that have been reported in the literature, yet very littleis known about the unique characteristics of the selectshRNAs and the pathway that they use to activate IFNproduction. The present study identifies RIG-I, but notMDA5 or TLR3, as the mediator for activation of IFNresponses by two shRNAs that are distinct in sequenceand structure but both capable of IFN induction inhuman cells. This was demonstrated by induction ofIRF-3 dimerization, activation of IFN promoters,induction of endogenous ISGs (ISG15, OAS andRIG-I), and secretion of IFN, all of which depended onRIG-I and its downstream adaptor, MAVS. In addition,we show that delivery of these shRNAs via lentiviraltransduction does not reduce their IFN-inducingcapacity, indicating that the ability of lentiviral vectortransduction to avoid IFN induction by shRNAs, asreported previously (48), may not be universallyapplicable to all the shRNAs.Specific recognition of dsRNAs or ssRNAs bearing

50-triphosphates by RIG-I is presumably determinedmostly by structural features other than the nucleotidesequence of the RNA. Yet IFN activation by sh-B971exhibited a stringent dependence on specific nucleotides

at multiple positions of the shRNA. An AA dinucleotideat the beginning of the U6 transcript has previously beensuggested to result in aberrant transcription, andpreserving a C/G sequence at positions �1/+1 suggestedto avert IFN induction (38). We indeed observed a strictrequirement for an adenylate at the +1 position of sh-B971 for RIG-I recognition and IFN activation, but weobserved no difference in expression levels or the apparentsizes of the sh-B971 RNAs bearing either an A or a G atthe +1 position. Furthermore, mutations introducedelsewhere in the shRNA also abolished or diminishedsh-B971’s ability to activate IFN, suggesting additionalsequence requirement for efficient RIG-I recognition andIFN triggering. Despite these results, because we werenot successfully in cloning and sequencing the vector-expressed siRNA, we cannot exclude the possibility thatthe adenylate at the +1 position interferes withtranscription and that the resultant abnormal transcriptcontributes to IFN induction.

Interestingly, the loop A mutant, which contains apredicted loop of 7 nt, generated a siRNA duplex insidethe cells that is slightly smaller than that of the shRNAswith a wild-type hairpin loop, suggesting the processing byDicer into the stem, perhaps fulfilling the requirement of alength of 9 nt for the hairpin loop (46). This mutant formof sh-B971 was not, however, able to trigger IFNactivation.

Despite the abilities of both sh-B971 and sh-PCAF toactivate the RIG-I pathway, the two shRNAs areunrelated in sequence. Two short stretches of siRNAsequences, GUCCUUCCAA and UGUGU, that havebeen previously defined as IFN- or cytokine-activatingmotifs (8,9) are not found in either sh-B971 or sh-PCAF. Any common sequence motifs of IFN-activatingshRNAs, if any, remain to be defined. The two shRNAsalso differ in that one is predicted to contain one blunt endand the other two ends with overhangs. These resultssuggest that, although blunt ends may increase siRNA’sability to be recognized by RIG-I (47), they are notrequired for IFN activation by an endogenously expressedshRNA. The best-characterized RNA structure motifrecognized by RIG-I is the 50-ppp, which is absent fromvirtually all the cellular RNAs as a result of either50-capping or internal cleavage before their appearancein the cytoplasm. A synthetic shRNA that has the samesequence as sh-B971 but lacks the 50-ppp failed to induceIFN, suggesting the 50-end status of the intracellularlyexpressed sh-B971 contributes to IFN activation.Whether or not the 50-end of an shRNA is capped hasnot been investigated. Murine U6 RNA does notcontain the trimethylguanosine cap that is present onmRNAs and other U small nuclear RNAs; instead itcontains a g-monomethyl phosphate cap at its 50-end(49). Capping of heterologous transcripts produced fromthe mU6 promoter, however, requires a stem loop at the50-end of the transcript and an AUAUAC sequenceimmediately after (50). Most shRNAs, including sh-B971and sh-PCAF, would not meet these requirements andthus should contain unmodified 50-ppp. Similarly, noevidence of a cap structure for H1 transcripts could befound in the literature. We attempted to express sh-B971



using a miRNA expression cassette and the pol IIpromoter (51). The primary transcript generated with thisconstruct would be capped at 50-end by a trimethyl-guanosine cap and the final siRNA duplex would bear amonophosphate at the 50-ends of both strands because ofDrosha and Dicer cleavage. This version of the sh-B971vector was much weaker in its ability to trigger IFNactivation. Unfortunately the intracellular expressionof the RNA duplex was also much weaker and barelydetectable by northern blotting. In addition, no knock-down of the target CyPB mRNA was seen with thismiRNA-based sh-B971 (data not shown). As a result,whether sh-B971, if expressed at higher level from thisconstruct, could effectively activate IFN remains unclear.

So far as we know, ours is the first report of IFNactivation in the target cells by shRNAs deliveredby lentiviral transduction. A previous report of IFNinduction by lentiviral vector-expressed shRNA onlyexamined the IFN generated in the vector-producingcells, which then up-regulated IFN-stimulated genes inthe transduced cells (10). The distinction is important aslentiviral vectors used in a gene-therapy setting will likelybe purified and free of any IFN that has been generatedduring the vector preparation step, but IFN activation inthe target cells would pose a more serious concern. Ourdata suggest the importance of screening shRNAs for IFNinduction in the transduced cells in vitro before large-scale studies. An HIV reverse transcriptase inhibitorefficiently blocked IFN production by both sh-B971 andsh-PCAF when delivered by transduction, indicating thevirion-encapsulated RNA was not able to trigger IFNactivation. In this respect, it is interesting to note thatpositive-stranded RNA viruses, which produce dsRNAintermediates in the cytoplasm during replication(52–55), often replicate in membrane enclosed vesicles(56), This sequestration of viral dsRNA in membranousstructures may shield the RNA from the cytoplasmicPRRs and contribute to a successful infection.

IFN-induction and RNAi by shRNAs appear to beindependent functions of the same RNA (57). Ourresults also showed that IFN-induction by sh-B971 isindependent of its ability to suppress target mRNAexpression through RNAi. On the other hand, it mightbe possible to screen for duel functional siRNAs thatconfer therapeutic benefits by both RNAi and immuno-stimulation (58). For example, siRNAs that target eitherviral genomes or cellular cofactors of the viruses can bescreened for their ability to trigger IFN activation in hopesof find ‘super siRNAs’ with increased efficacy againstIFN-sensitive viruses.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

The authors thank Dr Andre Irsigler and Dr JasonRobotham for technical assistance and Dr Anne B.Thistle for proofreading the manuscript.

FUNDING

National Institutes of Health (AI069285 to K.L.;DE016680 to F.Z.); and the Department of BiologicalScience at Florida State University (to H.T.). Fundingfor open access charge: The American Cancer Society.

Conflict of interest statement. None declared.

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Short-hairpin RNAs delivered by lentiviral vector transduction trigger